The development of anti-microbial agents necessitates the discrimination of the host organism from that of the invading pathogen. To achieve this end, unique features of the pathogen are logically targeted by the anti-microbial agent. There is an ongoing need for the identification and development of novel anti-microbial agents having specificity for the target pathogen, without exhibiting toxicity to the host organism.
Bacteriophage T4 development within E. coli is regulated by a controlled program of transcriptional activation and repression (Mosig & Hall, 1994, Molecular Biology of Bacteriophage T4, pp. 127–131; Sanson & Uzan, 1995, FEMS Microbiol. Rev. 17: 141–150). Within a few minutes after infection, the host RNA polymerase (RNAP) holoenzyme (Eσ70) transcribes several early genes of the bacteriophage genome that encode proteins capable of blocking host replication, transcription and translation. To redirect the host Eσ70 to transcribe T4 gene products, Eσ70 is modified through both covalent ADP-ribosylation of the α subunits and through binding of phage encoded proteins to the Eσ70 (Stitt & Hinton, 1994, Molecular Biology of Bacteriophage T4, pp. 142–160). Whereas expression of early T4 genes relies on unmodified polymerase, gene expression later in the life cycle requires a modified Eσ70 to effectively transcribe T4 genes.
AsiA, an early gene product, is necessary to ensure the progression of transcription from the T4 early to middle genes. AsiA is a small protein (10.6 kD) whose amino acid sequence is unrelated to any other protein in either the prokaryotic or eukaryotic sequence databases (Orsini et al., 1993, J. Bact. 175: 85–93). AsiA strongly represses transcription from host promoters in vitro (Stevens, 1976, RNA Polymerase, pp. 617–627; Orsini et. al., 1993, J. Bact. 175–85–93), although AsiA probably acts in tandem with other phage encoded factors to achieve complete repression of host gene expression in vivo (Pene & Uzan, 2000, Mol. Microbiol. 35: 1180–1191). AsiA additionally functions as a co-activator for phage middle gene expression in conjunction with the T4 MotA protein (Ouhammouch et al., 1995, PNAS 92: 1451–1455; Hinton, et al., 1996, Methods Enzymol. 274: 43–57). These dual regulatory events with opposing transcriptional consequences are the result of specific binding between AsiA and host Eσ70 wherein AsiA interacts with the C-terminal conserved domain of σ70 (termed region 4 (SR4)) (Severinova et al., 1996, J. Mol. Biol. 263: 637–647; Adelman, et al., 1997, J. Biol. Chem. 272: 27435–27443; Pahari & Chatterji, 1997, FEBS Letters 411: 60–62; Colland et al., 1998, Mol. Microbiol. 27: 819–829; Severinova et al., 1998, J. Mol. Biol. 279: 9–18; Sharma et al., 1999, J. Mol. Biol. 290: 905–915; Minakhin et al., 2001, J. Mol. Biol. 306: 631–642). Genetic and biochemical experiments have suggested that AsiA interacts with SR4 in a 1:1 complex (Adelman, et al., 1997, J. Biol. Chem. 272: 27435–27443). The interaction with SR4 most likely occurs with at least a portion of the putative DNA-recognition helix of SR4 (Pahari & Chatterji, 1997, FEBS Letters 411: 60–62; Colland et al., 1998, Mol. Micobiol. 27: 819–829; Severinova et al., 1998, J. Mol. Biol. 279: 9–18; Minakhin et al, 2001, J. Mol. Biol. 306: 631–642). AsiA is capable of tightly associating with SR4 in the presence or absence of core RNAP (Severinova et al., 1996, J. Mol. Biol. 263: 637–647; Adelman, et al., 1997, J. Biol. Chem. 272: 27435–27443; Pahari & Chatterji, 1997, FEBS Letters 411: 60–62; Colland et al., 1998, Mol. Microbiol. 27: 819–829; Severinova et al., 1998, J. Mol. Biol. 279: 9–18) and there may be a preferential association with free σ70 in order to establish the full repressive effect of AsiA on host gene expression (Hinton & Vuthoori, 2000, J. Mol. Biol. 304: 731–739). The consequence of AsiA interaction with SR4 is to block the formation of closed Eσ70 complexes at host promoters which harbor the two conserved σ70 DNA binding elements centered at −10 and −35. At T4 middle promoters, the σ70 −35 binding element is replaced with a binding site for MotA centered at −30. The presence of a MotA/DNA complex preferentially recruits AsiA-modified Eσ70 to T4 middle promoters to specifically transcribe these genes.
It is toward a better understanding of modeling and identification of anti-sigma factor agents of various bacterial species that the present invention is directed, based upon the precise definition acquired from the nature of the interactions between AsiA and the SR4 domain of Eσ70 as derived from the studies herein.